Porphyromonas gingivalis lipopolysaccharide (LPS). The primary hG-MSCs were treated with titanium and zirconia nanoparticles in concentrations up to 2.000 µg/mL for 24 h, 72 h, and 168 h. Additionally, the cells were treated with different nanoparticles (25–100 µg/mL) in the presence of P. gingivalis LPS for 24 h. The cell proliferation and viability assay and live–dead and focal adhesion stainings were performed, and the expression levels of interleukin (IL)-6, IL-8, and monocyte chemoattractant protein (MCP)-1 were measured. The cell proliferation and viability were inhibited by the titanium (>1000 µg/mL) but not the zirconia nanoparticles, which was accompanied by enhanced apoptosis. Both types of nanoparticles (>25 µg/mL) induced the significant expression of IL-8 in gingival MSCs, and a slightly higher effect was observed for titanium nanoparticles. Both nanoparticles substantially enhanced the P. gingivalis LPS-induced IL-8 production; a higher effect was observed for zirconia nanoparticles. The production of inflammatory mediators by hG-MSCs is affected by the nanoparticles. This effect depends on the nanoparticle material and the presence of inflammatory stimuli. Nano- and microparticles are currently being discussed as potential risk factors for peri-implant disease. In the present study, we compared the responses of human gingival mesenchymal stromal cells (hG-MSCs) on titanium and zirconia nanoparticles (<100 nm) in the absence and presence of  Dental implants have been proven to be secure and long-lasting options for replacing missing teeth, providing a pivotal treatment modality in modern dentistry ,2. The bOsseointegration is defined as the rigid fixation of an implant in the surrounding bone, and is pivotal for the clinical outcome and long-term stability . ImplantAlthough modern dental implants demonstrate a high success rate, some implant failures still occur. The major reason for implant loss is peri-implantitis, which is characterized by the inflammation of soft and hard peri-implant tissues . The preMaterial particles can be released during the implant placement  via corrPorphyromonas gingivalis lipopolysaccharide (LPS), because this putative periodontal pathogen is often found in peri-implant lesions [Human gingival mesenchymal stromal cells (hG-MSCs) are one of the major constituents of peri-implant soft tissue and play an important role in the production of the extracellular matrix and the inflammatory response ,29. Prev lesions .The proliferation and viability rates of the hG-MSCs after the treatment with Ti and Tz NPs for 24 h, 72 h, and 168 h are presented in The live–dead stainings of hG-MSCs cultured in the presence or in the absence of different NPs at a concentration of 1000 µg/mL are presented in The effects of different NPs at concentrations of 250 and 1000 µg/mL on the basal production of IL-6, IL-8, and MCP-1 in hG-MSCs after 24 h, 72 h, and 168 h are shown in Both types of NPs significantly enhanced the gene expression of IL-8 at all concentrations throughout the whole observation period . After 7No significant effect of any NP on the gene or protein expression levels of MCP-1 was observed . After 7P. gingivalis LPS-induced response in hG-MSCs after 24 h of incubation are shown in P. gingivalis LPS significantly increased the gene expression levels and protein production rates of IL-6, IL-8, and MCP-1. The NPs did not affect the P. gingivalis LPS-induced IL-6 gene expression or protein production. The P. gingivalis LPS-induced IL-8 gene expression was significantly enhanced by the Zr NPs at a concentration of 100 µg/mL, and the IL-8 protein production was enhanced by the Zr NPs at concentrations of 25 and 100 µg/mL and by Ti NPs at a concentration of 100 µg/mL. Additionally, in the presence of P. gingivalis LPS, the Zr NPs induced significantly higher IL-8 protein production rates than Ti NPs at the NP concentration of 100 µg/mL. The P. gingivalis LPS-induced MCP-1 gene expression was significantly inhibited by the NPs, except for the Ti NPs at a concentration of 25 µg/mL. Under these conditions, the gene expression of MCP-1 in the presence of the Zr NPs was significantly lower than those in the presence of the Ti NPs. No significant effect of the NPs on the MCP-1 protein production in the presence of P. gingivalis LPS was observed.The effects of various NPs on the In the present study, we focused on a comparison of the effects of Zr and Ti NPs on primary hG-MSCs. Our data showed that Ti particles exhibit higher cytotoxicity toward hG-MSCs than Zr particles. This was suggested by the finding that the proliferation or viability of hG-MSCs was inhibited by the Ti particles starting from the concentration of 1000 µg/mL, whereas no cytotoxic effect of Zr particles was observed. A higher cytotoxicity of Ti NPs was also confirmed by the live–dead staining. These data are in agreement with the previous data with particles of a similar size. In particular, the cytotoxicity of the Ti nanoparticles in immortalized human periodontal ligament cells (EC50 2.8 mg/mL) was up to five times higher than that of Zr nanoparticles (EC50 13.96 mg/mL) . The reaThe analysis of the cell morphology showed that the presence of nanoparticles resulted in an altered cell morphology. The presence of nanoparticles or their conglomerates provided some additional attachment places for the cells, leading to the different morphologies. This assumption was confirmed by the re-distribution of vinculin staining in the presence of nanoparticles . Under tP. gingivalis LPS. In the absence of P. gingivalis LPS, only rather high concentrations of nanoparticles (>250 ng/mL) were able to upregulate the production of pro-inflammatory mediators via hG-MSCs. Some differences were observed between the nanoparticles of different materials. Interestingly, at the gene level, in many cases the expression level of pro-inflammatory mediators in hG-MSCs was higher after the treatment with Ti compared to the zirconia nanoparticles. However, these findings were rarely confirmed by the protein data, and even an opposite tendency was observed. Thus, the Zr nanoparticles induced significantly higher IL-6 and IL-8 protein production levels after 24 h and greater MCP-1 production after 72 h than Ti NPs, despite the similar gene expression levels. Moreover, 1000 µg/mL of Zr NPs induced a significantly higher IL-8 protein concentration than the Ti NPs, although at the gene level an opposite effect was observed. The lacking correspondence between the gene expression and protein data could be partially due to the cytotoxic effect of the Ti NPs, which can lead to a lower number of functionally active cells and to lower protein production.We further investigated the effects of various nanoparticles on the inflammatory response of the hG-MSCs. This was done in the presence and in the absence of Previous studies differently described the ability of material particles to induce the inflammatory response in different host cells. In gingival fibroblasts, a low concentration of Ti particles (in the range of µg/mL) induced the production of IL-6 . In fibrP. gingivalis LPS, which was used to mimic the inflammatory environment characteristics for the peri-implantitis. We found that the NP-modified P. gingivalis induced an inflammatory response depending on the cytokine type and NP material. Thus, both types of NPs enhanced the P. gingivalis LPS-induced Il-8 production, and this effect was slightly higher for Zr NPs than Ti NPs. In contrast, the MCP-1 expression was reduced by both NPs, and higher inhibition was observed for Zr than Ti NPs. IL-8 is a potent chemokine for polymorphonuclear neutrophils, and MCP-1 stimulates monocyte migration. A stimulating effect of Ti particles measuring over 0.22 µm on LPS-induced IL-1β production was previously reported in THP-1 macrophages [The material NPs also modified the response of hG-MSCs to rophages . The traIn our study, we focused on the particles with a size of about 100 nm. The particles of this size are released during implantoplasty , which iThe role of material particles in the etiology of peri-implantitis is debatable to date ,45,46. TZirconia seems to be superior to Ti in the terms of the release of particles and their adverse effects in gingival tissue. Our data and other studies show that the toxic effect of material particles is observed for Zr at higher concentrations than for Ti. Furthermore, zirconia as a non-metal is assumed to release fewer particles than titanium. This statement is supported by one study showing that platform-switched constructs consisting of a Ti implant and Zr abutment released up to 10 times less particles than platform-matched constructs consisting of Ti upon the application of static forces and an acidic environment . A recenSome of the effects of NPs in our study were observed at physiologically relevant concentrations. There have been different estimations about the amount of material particles in peri-implant tissue. Thus, a study on a mini pig reported up to 2.17 mg/kg of bone Ti and 0.59 mg/kg of bone zirconia around Ti and Zr implants placed in the maxilla , which cThe main limitation of the present study is the in vitro design. We used the cells isolated from systemically and periodontally healthy young individuals, and this population does not reflect peri-implantitis patients. We used only one cells type, whereas the inflammatory response in peri-implantitis involves various cells. In our study, we investigated only nanoparticles, but implant release particles of different sizes. Although NPs exhibit the most toxic effects on the host cells, the contribution of microparticles to the host response should be considered. Finally, through the interpretation of the effects of Ti and Zr, the differences in release rate and size between these materials should be considered.Ethical approval for the hG-MSC isolation was obtained from the Ethics Committee of the Medical University of Vienna, Vienna, Austria (vote no.1079_2019). Written and informed patient consent was given prior to tooth donations. The present study was conducted in compliance with the Declaration of Helsinki regarding the ethical principles for medical research involving human subjects and according to the Good Scientific Practice Guidelines of the Medical University of Vienna.2, and 95% humidity. The hG-MSCs were passaged after reaching 80% to 90% confluence and the medium was changed every three days. The cells between passages four and six were used for all experiments.Primary hG-MSCs were isolated from the third molar teeth of periodontally healthy individuals aged between 18 and 32 years, which were extracted due to orthodontic indications. The donors were periodontally and systemically healthy and did not take any regular medication three months prior to the study. The cells were isolated using the outgrowth method ,53. The 2, Ti NPs) nanopowder and zirconium (IV) dioxide  nanopowder, each with a particle size of <100 nm, were used. The particles were suspended in DMEM, supplemented with Pen-Strep without any FBS, to obtain a 2000 µg/mL stock solution.Two different commercially available  nanoparticles, titanium (IV)dioxide  and 200 ng/mL soluble CD14  [Here, 5 × 10is, USA) . For theThe cell viability and proliferation were evaluated after 24 h, 72 h, and 168 h of stimulation using 3--2,5-diphenyltetrazoliumbromid  as described previously . In brie2, and 95% humidity. Afterwards, the fluorescence staining was directly visualized using the EchoRevolve fluorescence microscope , distinguishing between calcein-AM  and propidium iodide (Ex/Em = 570/602 nm).The live–dead analysis was performed after 24 h, 72 h, and 168 h of incubation using the Live/Dead Cell Staining Kit from Enzo Life Sciences  following the manufacturer’s guidelines. Briefly, after washing the cells with 1 × PBS, 150 µL of staining solution containing calcein-AM and propidium iodide for staining live and dead cells, respectively, was added per well followed by incubation for 15 min at 37 °C, 5% COThe focal adhesions were stained after 24 h, 72 h, and 168 h of incubation for hG-MSCs using the Actin Cytoskeleton/Focal Adhesion Staining Kit from Merck , according to the manufacturer’s instructions. In brief, after fixing and permeabilizing the cells with 4% paraformaldehyde and 0.1% Trition X-100 in 1 × PBS, respectively, the hG-MSCs were blocked by adding 1% BSA in 1 × PBS. The actin filaments were stained with TRITC-conjugated anti-phalloidin (Ex/Em 540/565 nM) for focal adhesion with the anti-vinculin monoclonal antibody (purified clone 7F9) followed by the FITC-conjugated secondary antibody (Ex/Em 495/520 nm). This was followed by nuclei counterstaining with 4′,6-diamidin-2-phenylindol . Brightfield and fluorescence pictures were taken using the EchoRevolve fluorescence microscope .P. gingivalis LPS based on the production of the pro-inflammatory mediators IL-6, IL-8, and MCP-1. In the absence of P. gingivalis LPS, the inflammatory response was evaluated after 24 h, 72 h, and 168 h, whereas in the presence of LPS it was done after 24 h.The effects of different nanoparticles on the production of inflammatory mediators by hG-MSCs was assessed in the absence or in the presence of ® Gene Expression Cells-to-CT™ kit  according to the manufacturer’s instructions. The reverse transcription was performed using a Primus 96 advanced thermocycler  by heating the samples to 37 °C for one hour followed by 95 °C for five minutes. Here, qPCR was performed on an ABI StepOnePlus device  in paired reactions using TaqMan® gene expression assays with the following ID numbers : interleukin-8 (IL-8) (Hs00174103_m1), interleukin-6 (IL-6) (Hs00985639_m1), monocyte chemoattractant protein (MCP-1) (Hs00234140_m1); glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Hs99999905_m1). GAPDH was used as a house-keeping gene. The PCR reactions were performed in triplicate under the following conditions: 95 °C for 10 min, followed by 50 cycles, each for 15 s at 95 °C and at 60 °C for 1 min. For each sample, the point at which the PCR product was first detected above a fixed threshold  was determined. The 2−ΔΔCt method was used to calculate the relative expression of target genes by taking unstimulated hG-MSCs as a control; here, ΔΔCt was calculated using the following formula:The gene expression levels were quantified using qPCR, similarly to our previously published study . Cell lyThe levels of IL-6, IL-8, and MCP-1 proteins in conditioned media were determined using a commercially available ELISA  according to the manufacturer’s instructions. The conditioned media were harvested and centrifuged to remove the cell debris and nanoparticles. For all ELISAs, the OD values were measured at 450 nm and 570 nm using a Synergy HTX multi-mode reader  and their concentrations in the conditioned media were calculated based on the standard curves ranging 3.125–200 pg/mL for IL-6, 3.9–250 pg/mL for IL-8, and 15.6–1000 pg/mL for MCP-1. All values below the detection limit were considered as zero for the analysis.p values less than 0.05 were considered to be statistically significant. The data are presented as means ± standard errors of the mean. All statistical analyses were performed using SPSS 24.0 software . The evaluation of LDS and FAK images was performed qualitatively.All experiments were repeated at least five times, with cells isolated from five different donors. Each experiment was performed at least in technical triplicate. Due to the low number of experiments, it was not possible to confirm the normal distribution, meaning non-parametric tests were used. The differences between groups were assessed using the Wilcoxon signed-rank test. Here, The NPs released from the implant material influence the functional activity of the primary hG-MSCs under normal and inflammatory conditions. Our data show that at similar concentrations, the Ti NPs have a more detrimental effect on the hG-MSCs compared to the Zr NPs. Taking into consideration the generally lower release of particles by Zr than Ti, our data suggest that Zr implants could be superior in terms of the potential side effects due to particle release.


NRAS gene is a well-known oncogene that acts as a major player in carcinogenesis. Mutations in the NRAS gene have been linked to multiple types of human tumors. Therefore, the identification of the most deleterious single nucleotide polymorphisms (SNPs) in the NRAS gene is necessary to understand the key factors of tumor pathogenesis and therapy. We aimed to retrieve NRAS missense SNPs and analyze them comprehensively using sequence and structure approaches to determine the most deleterious SNPs that could increase the risk of carcinogenesis. We also adopted structural biology methods and docking tools to investigate the behavior of the filtered SNPs. After retrieving missense SNPs and analyzing them using six in silico tools, 17 mutations were found to be the most deleterious mutations in NRAS. All SNPs except S145L were found to decrease NRAS stability, and all SNPs were found on highly conserved residues and important functional domains, except R164C. In addition, all mutations except G60E and S145L showed a higher binding affinity to GTP, implicating an increase in malignancy tendency. As a consequence, all other 14 mutations were expected to increase the risk of carcinogenesis, with 5 mutations  expected to have the highest risk. Thermodynamic stability was ensured for these SNP models through molecular dynamics simulation based on trajectory analysis. Free binding affinity toward the natural substrate, GTP, was higher for these models as compared to the native NRAS protein. The Gly13 SNP proteins depict a differential conformational state that could favor nucleotide exchange and catalytic potentiality. A further application of experimental methods with all these 14 mutations could reveal new insights into the pathogenesis and management of different types of tumors.The  Single nucleotide polymorphism (SNP) is defined as a variation in one base in a DNA nucleotide that happens at a specific site of the studied genome . In the HRAS, KRAS, and NRAS, are members of the Ras family with GTPase activity. These genes function as molecular switches in the cell, where Ras GTP represents the active state, while Ras GDP represents the inactive state  to retrieve gene ontology data and (compartments.jensenlab.org) as the source of data regarding the subcellular localization.We depended on the National Center for Biotechnology Information (NCBI) databases besides Ensembl databases for retrieving general information regarding the NRAS gene variants using variation viewer and by selecting dbSNP as the source database (https://www.ncbi.nlm.nih.gov/variation/view/). “NRAS” or 4,893 [geneid] was used as an entry. We filtered those retrieved SNPs and selected only the missense variants for further analysis and screening.NCBI was used to retrieve in silico tools for predicting the most deleterious variants on the function of NRAS protein: SIFT (Sorting Intolerant from Tolerant), PolyPhen (Polymorphism Phenotyping), PROVEAN , SNAP, SNP&GO, and PHD-SNP. The SNPs that were predicted to be deleterious by all tools were considered the most deleterious ones. Using six different tools with varying algorithms, an approach that was applied previously  (http://genetics.bwh.harvard.edu/pph2) (http://provean.jcvi.org/seq_submit.php) (https://snps.biofold.org/snpsand-go/snps-and-go.html) (http://snps.biofold.org/phd-snp/phd-snp.html) (https://rostlab.org/services/snap/)  . PolyPhedu/pph2) . The PROmit.php) . The SNPgo.html) . The PHDnp.html) . SNAP2 hs/snap/) .https://folding.biofold.org/i-mutant/i-mutant2.0.html) relies on a support vector machine for the prediction of the direction and the value of the free energy change (DDG) (http://mupro.proteomics.ics.uci.edu/) provides a support vector machines method that was tested and cross-validated with an accuracy of 84%  . I-Mutange (DDG) . The Mu-y of 84% .https://www.ebi.ac.uk/interpro/). InterPro could perform the functional analysis related to the nominated protein and determine its functional domains and important motifs (We used InterPro to determine the locations of the most deleterious variants on the domains of NRAS protein (t motifs .https://consurf.tau.ac.il). ConSurf can analyze the phylogenetic relationships that exist between various homologous sequences to perform the needed analysis with the calculation of the conservation score that ranges from one to nine. Furthermore, the structural and functional residues are identified as well  for identifying the predicted secondary structures of NRAS protein and the alignment of the mutant amino acids in these secondary structures. The SOPMA server can predict the secondary structures of a selected protein by analyzing the multiple alignments of this protein’s sequence  was employed to estimate the TM-score and root-mean-square deviation (RMSD) values of the parent model selected by SWISS-MODEL and the mutant ones that were predicted. The TM-score gives information about the degree of similarity between submitted structures, and its value ranges between 0 and 1 . On the other hand, the RMSD value indicates the average distance between alpha-carbon backbones of analyzed models, and the higher this value is, the more deviation is predicted between analyzed molecules , leading to overactive cellular signaling and ultimately cancer. The NRAS wild tertiary structure (PDB ID: 5UHV) was downloaded in PDB format to act as a control receptor for the current docking study; the deposited molecules in the receptor were first removed and the generated molecule saved to act as a control for its binding score with GTP to validate the docking scores of GTP-binding with other mutated models. The docking was performed through AutoDock Vina  and GTP molecule (the ligand of NRAS active state) was proposed. As mentioned in the introduction section, NRAS is a GTP-binding protein that is switched on by incoming signals, leading to a turn-on for genes related to cell growth and differentiation. Therefore, SNPs in ock Vina , the 3D ock Vina , and a 2ock Vina .2). Free binding energies for GTP substrate binding to GTPase NRAS were estimated using Molecular Mechanics/Poisson-Boltzmann  single trajectory calculations  by applying the elastic torsional network model, whose degrees of freedom are the protein backbone torsion angles (The collective flexibilities/motion functions of the constructed GTPase NRAS were assessed using the iMODS online server (n angles . This aphttps://www3.cmbi.umcn.nl/hope/) for analyzing the SNPs’ impact on the NRAS protein 3D structure. The HOPE server depends on several sources for collecting the needed information in addition to building homology models using the YASARA program to perform the required function (We used the HOPE server (function .https://www.musite.net) was used to predict the sites of different types of PTMs. PTMs have a major role in regulating the function of proteins; thus, the identification of PTMs is important in analyzing disease pathogenesis (The MusiteDeep server (ogenesis .http://www.genemania.org) for generating the gene–gene interaction network of the NRAS gene. The GeneMANIA tool could predict the strongly interacted genes with a selected gene using various sorts of data and resources: co-expression information, physical interaction information, co-localization data, functional relationships, information regarding pathways, genetic interaction data, and data regarding protein domains  is a protein-coding gene that is located at 1p13.2. It comprises seven exons and has a length of 12,303 nucleotides; it has one transcript (ensemble.org). The membrane protein encoded by this gene shuttles between the plasma membrane and the Golgi apparatus and is characterized by GTPase activity (https://www.ncbi.nlm.nih.gov/gene/4893). The subcellular localization of the NRAS gene is illustrated in Genecards.org).The NRAS gene, 4,542 single nucleotide variations were found (accessed 31 December 2021). Among these SNPs, there were 113 missense SNPs, 62 synonymous SNPs, 73 5′ untranslated region (UTR) variants, 1,405 3′ UTR variants, and 2,435 intron SNPs, in addition to the other downstream and upstream SNPs.In the NRAS gene with a significant impact on NRAS protein function. Of note, 16 variants were found to be disease-causing and deleterious by all six tools, and the percentage of each SNP type is shown in Six different bioinformatics tools  were used to determine the deleterious variants in the The impacts of SNPs on NRAS protein stability were analyzed using the I-Mutant 2.0 server and the Mu-Pro tool. The I-Mutant 2.0 server found that 12 SNPs had a decreasing effect on NRAS protein stability, while the Mu-Pro tool showed a decreasing effect with all SNPs except rs1163400692 SNP. The predicted impacts and values are shown in The InterPro tool analyzed NRAS protein functionally and revealed the presence of an important domain: a small GTP-binding protein domain (InterPro entry: IPR005225). The positions of the 16 deleterious variants were analyzed, and all SNPs were found to be located in this domain, except for rs1658971260, as shown in The evolutionary conservation of NRAS protein amino acids was analyzed as displayed in The predicted secondary structures of NRAS were analyzed via the SOPMA tool as displayed in To predict the tertiary structure of the models with 17 different mutations, SWISS-MODEL presented the wild-type NRAS 3D model with PDB ID of 5UHV as the best choice for homology modeling with 99.39% identity with all the mutated submitted sequences. Validation of these models was performed by detecting the ProSA Z score and the percentage of residues in the favored region for each molecule , where tThe current docking study was performed to demonstrate the difference in the binding affinity between native and mutated models with the GTP ligand. The binding score between the native NRAS and GTP was −11.3 kcal/mol, and for the mutant predicted structures, the respective binding score is shown in The 3D complexes of the native model, model #1 (the one with the highest binding affinity), and model #11 (the one with the lowest binding affinity) with GTP are visualized by PyMOL, shown in Simulated proteins of the GTPase NRAS models showed steady equilibrated RMSD trajectories, respective to initial proteins’ alpha-carbons, across the 100-ns runs . The ste2, 15.24 ± 0.07 Å//89.64 ± 2.25 nm2, and 15.19 ± 0.05 Å//86.69 ± 1.74 nm2, were assigned for the simulated model numbers 2, 6, and 16, respectively. Again, the native holo NRAS protein showed a slight increase in its Rg and SASA tones, which then proceeded constantly till the end of the simulation runs. Similar to the protein’s RMSDs, the monitored SASA trajectories were of the highest values for the simulated apo native NRAS protein as compared to other simulated congerants.Monitoring the RMSD trajectories of the GTP ligand, in relation to its initial/reference position, showed steady tones for the simulated models . The RMSApo—RMSFHolo) was of better representation for the residue-wise protein stability and fluctuation. Residues depicting ΔRMSF values ≥ 0.3 cut-offs were considered significantly rigid, showing negligible mobility  depicted a significant immobility profile with ΔRMSF above the 0.30 Å cut-off value. Notably, stability residue-wise ranges were more prominent for the effector lobe rather than those at the allosteric half of the GTPase catalytic domain. Concerning the comparative NRAS flexibility profiles, model #3, along with the native holo protein, showed the highest mobility tones with more negative and less positive ΔRMSF values. It is interesting that SNP NRAS models #1, #2, and #3 depicted consistent mobility and flexibility profiles for the 37–50 residue range, showing almost all its respective residues with negative ΔRMSF values. Moreover, selected switch-II residues  exhibited high ΔRMSF values for the same three Gly13 SNP models. It is worth noting that the native holo protein depicted negative ΔRMSF values for the same previous switch-I and -II residue ranges yet at less prominent negative numbers. On different bases, the three Gly13 SNP residues, G13R, G13C, and G13V, in models #1, #2, and #3, respectively, showed a slightly higher immobility profile (ΔRMSF ∼0.60 Å) than their native Gly13 in holo NRAS protein. The P34R SNP residue in model #6 showed a more preferential stability profile than its native Pro34 congerant. Likewise, the V152F SNP in model #16 was more rigid, reaching a positive ΔRMSF value (0.24 Å), as compared to its negative-valued native residue .Conformational analysis for the simulated NRAS was performed to grasp the main secondary structure changes and SNP residue conformational shift throughout the molecular dynamics simulations. Trajectories at the start and end of the simulation runs were extracted, minimized at 0.0001, and finally overlayed. As expected, the docked GTP molecule was attained within the NRAS-binding site at the end of molecular dynamics simulation runs . Most SNIt is worth mentioning that significant secondary structure conformational alterations were also depicted and such observations were differential across each simulated model. Regarding the G13x SNP model, a significant shift of the switch-I loop of the simulated NRAS proteins at the end of the molecular simulation runs. A switch from the closed state-1 to an open state-2 was depicted with models #1–3, being more obvious for model #1. This switch-I drift was accompanied by an outward movement for the α-helix 3 and loop 7 away from the switch-II domain. The latter drift permitted the switch-II to attain the catalytically active R-state conformation rather than the catalytically incompetent T-state at the start of simulation runs. The above-described switch-I and switch-II movements were less prominent with SNP models #6 and #16, where minimal or insignificant conformational changes were depicted. The native holo NRAS complex exhibited moderate switch-I drift toward the open state-1, yet without significant outward movement of its α-helix 3/loop 7 nor its switch-II domain.Estimating the free binding affinity of GTP toward the constructed NRAS models as well as the native holo state was performed using the trajectory-oriented MM-PBSA calculations. Using the single trajectory approach and SASA-only model , the total binding energies for GTP in all SNP NRAS models were superior to that of the native holo protein . This SNThe obtained total binding-free energies were further decomposed down to the residue-wise binding energy contributions  to identSNP model #1: Arg13-side (30.64%), Lys16-side (93.41%), Ala18-main (44.31%), Asp30-main (30.34%), Asp119-side (123.05%); SNP model #2: Cys13-side/main (22.45%), Lys16-side (69.06%), Ala18-main (14.22%), Asp30-side (50.82%), Asp119-side (118.66%); SNP model #3: Lys16-side/main (107.29%), Ala18-main (52.10%), Asp119-side (123.45%); SNP model #6: Gly13-main (30.84%), Lys16-side/main (131.62%), Ala18-main (34.73%), Asp30-main (32.63%), Tyr32-side (53.29%), Arg34-side (38.82%), Asp119-side (123.25%); SNP model #16: Lys16-side/main (132.23%), Tyr32-side (63.97%), Asp119-side (115.87%); and native holo NRAS: Lys16-side (105.39%), Ala18-main (41.72%), Asp30-main (35.53%), Asp119-side (120.86%).Most of the latter top-energy-contributing polar residues showed high hydrogen bond % frequencies with GTP throughout the entire simulation runs using the VMD hydrogen–bond tools. On the contrary, other hydrophilic pocket-lining residues, including Glu37, Arg38, Glu62, Glu63, and/or Asp69, exhibited unfavored positive-valued energies. The latter binding repellent residues were of the highest values for SNP models #1 and #6. Concerning the SNP residues, G13R showed a higher negative energy contribution in model #1 as compared to its SNP or native congerants. Likewise, model #6 showed significant binding energy for its SNP P34R, with a value reaching up to −113.0 kJ/mol. The SNP V152F residue showed relevant energy contribution in model #16, whereas the native congerant illustrated either poor energy contributions (∼−0.45 kJ/mol) or even positive-value energies (∼0.14 kJ/mol).−04) as compared to those of other SNP models as well as the native NRAS protein  showed the strongest relatedness with a rank of 1, followed by the STK19 (serine/threonine kinase 19) gene and the RGS12  gene consecutively.The GeneMANIA tool was used for generating the NRAS gene is a well-known oncogene with a major role in carcinogenesis . In total, 16 SNPs were predicted to be deleterious and disease-causing by all used tools. Due to the critical role of protein stability in protein function and structure  are expected to have the highest risk of carcinogenesis. It is important to mention that some of the nominated deleterious SNPs in the current study have already been correlated to carcinogenesis in previous wet-lab studies, where G13 mutations have been associated with chronic myelomonocytic leukemia and juvenile myelomonocytic leukemia progression in humans and mice  and G13VNRAS mutations have been linked to several forms of tumors in many previous studies, the current analysis would represent a guide for future experimental SNP validation and clinical analysis on a large group of people.The NRAS protein has a vital role in regulating cell proliferation, motility, and apoptosis. It has a domain that interacts with a GTP molecule and converts it to GDP to control the cell activity status. Hence, the conservation and structural conformation of this domain are highly important for the protein functional role. The current computational study sheds light on the deleterious effects of the nsSNPs on the structural and functional characteristics of this protein. Our study demonstrated that 14 SNPs are predicted to affect NRAS structure and function, where the 5 mutations that showed the highest binding affinity to GTP  were nominated as the highest risky ones. Thermodynamic stability was assured for these top-docked SNP proteins through molecular dynamics simulations. Furthermore, the Gly13 SNP model adopted differential conformational states that could confer significant GTP catalysis and nucleotide exchange. As


Mucor circinelloides produces a high lipid content (36%), including valuable polyunsaturated fatty acids (PUFAs). However, the critical step in which oleic acid (OA) is converted into LA is not efficient at supplying enough substrates for PUFA synthesis. Hence, we propose a method to increase LA production based on genetic engineering. The overexpression of the Δ12-desaturase gene from M. circinelloides and Mortierella alpina increased the LA content and improved the lipid accumulation (from 14.9% to 21.6% in the Δ12-desaturase gene of the M. circinelloides overexpressing strain (Mc-D12MC) and from 14.9% to 18.7% in the Δ12-desaturase gene of M. alpina overexpressing strain (Mc-D12MA)). Additionally, the up-regulated expression levels of these genes targeted the genes involved in NADPH production, implying that the elevated Δ12-desaturase gene may function as a critical regulator of NADPH and lipid synthesis in M. circinelloides. This study provides the first evidence to support the design of metabolic engineering related to LA and PUFA production in M. circinelloides for potential industrial applications.Linolenic acid (LA) is gaining more interest within the scientific community. This is because it has a potential medical role in reducing the risk of inflammation, carcinogenesis, atherosclerosis and diabetes and is a valuable nutraceutical for human health. The oleaginous fungus  Polyunsaturated fatty acids (PUFAs) have essential structural and functional roles in the human body. They are responsible for cell membrane fluidity and protein activity or interactions with other molecules . In addi+-dependent isocitrate dehydrogenase (NADP+: ICDH) [For the fatty acid biosynthesis pathway, oleaginous microorganisms require acetyl-CoA as a substrate generated via cytosol citrate cleavage and catalyzed by ACL (ATP: citrate lyase) . In micr+: ICDH) . PUFAs a+: ICDH) . Thus, L+: ICDH) .Mucor circinelloides was used as a commercial strain (in which the lipid content is 25% of the cell dry weight (CDW) for GLA production in the 1980s [M. circinelloides WJ11, isolated from soil, presented 36% of its own CDW [ own CDW , utilizi own CDW , and gly own CDW ).Mucor circinelloides can be genetically modified to increase the lipid content and the LA yield [Schwanniomyces occidentalis by homologously and heterologously expressing FAD2. Subsequently, Δ12-fatty acid desaturase was successfully expressed in Rhodotorula toruloides and improved the final titer of LA to 1.3 g/L [M. circinelloides, since there is only one Δ12-desaturase gene in its genome, and the LA content is approximately 15 % of the total lipids [M. alpina and M. circinelloides [M. circinelloides. The present study has the potential to improve the commercial value of microbial lipids and could provide a novel and sustainable source of LA-rich lipids.LA yield ,15,16. LLA yield  increase 1.3 g/L . LA is al lipids . Thus, telloides . To inveM. alpina (ATCC32222) and M. circinelloides WJ11 (CCTCC No. M 2014424) were amplified from the cDNA libraries of these two strains [M. circinelloides MU760, an uracil and leucine auxotrophic strain of M. circinelloides WJ11, was used as the recipient strain for Δ12-desaturase genes. During the genetic manipulations, transformants were selected in the minimal culture medium MMC and in MMC with the uracil medium, as previously described [6 spores of each strain were added to 100 mL of K & R medium in 500 mL baffled flasks and cultured in a shaker at 28 °C for 1 day, and then transferred all the cultures into a fermenter that contained 1 L K & R medium. The fermentation conditions were set at a stirring speed of 600 rpm, aeration of 1.0 min−1, temperature maintained at 28 °C, and pH adjusted to 6.0 using 1 mol/L NaOH.The Δ12-desaturase genes from  strains . M. circescribed . The KenEscherichia coli Top 10 (for cloning and plasmid storage) was cultured in an LB medium supplemented with ampicillin or kanamycin (100 mg/L) when necessary [The ecessary .pyrF) and a strong promoter (pzrt1) surrounded by 1 kb up-/down-stream CarRP sequences was used to insert the target genes. The Δ12-desaturase gene fragments were amplified from the cDNA libraries of M. alpina and M. circinelloides by the primers D12MA-F/R and D12MC-F/R  [To analyze the impacts of Δ12-desaturase gene overexpression on the fermentation process of , China) .2O, and freeze-dried for biomass and lipid analysis. For lipid and fatty acid analysis, 10 mg of freeze-dried mycelium were used for extraction using chloroform/methanol . Pentadecanoic acid (C15:0) was added to the extract as an internal standard, and the mixture was methylated by using 10% HCL/methanol (w/w). The fatty acid methyl esters (FAMEs) were analyzed by gas chromatography (GC) equipped with a 30 m × 0.32 mm DB-Waxetr column [Fungal mycelia were harvested by a Buchner funnel filtration, washed three times by distilled Hr column . The GC −ΔΔCt method was applied to calculate the gene expression levels. The primers used in this study are listed in the Transformants’ genomic DNA was extracted using the DNA Quick Plant System kit . Total RNA was extracted with Trizol and reverse transcribed to cDNA library using Evo M-MLV RT Mix Kit with gDNA Clean for qPCR . Quantitative reverse transcription-PCR analysis (RT-qPCR) was performed using SYBR Green Realtime PCR Master Mix in a LightCycler 96 . Actin was used as an internal reference gene, and the 2t-test  was used for statistical analysis of the data, and differences were considered statistically significant at p < 0.05.All of the experimental results were calculated as the mean data from three independent experiments, and the data were presented as mean ± SD. The student’s M. alpina and M. circinelloides, two genes (Δ12Mc and Δ12Ma) were cloned into the expression vector pMAT2075 individually and generated gene overexpressing plasmids pMAT2075-D12MC and pMAT2075-D12MA. The transformants (named Mc-D12MC and Mc-D12MA) were constructed by homologous recombination of the linearized fragments that contained the selectable marker pyrF gene, the target gene, and the genes flanking 1 kb up- and down-stream from the carRP gene. The control strain Mc-CS was generated by the fragments in pMAT2075  was 2.5-fold higher than that in the control strain, which suggested that Δ12Mc was successfully overexpressed in Mc-D12MC. Meanwhile, Δ12Ma only showed a significant expression level in Mc-D12MA, indicating that Δ12Ma was successfully overexpressed in M. circinelloides without affecting the native Δ12Mc expression pattern.The transcriptional levels of the Δ12-desaturase gene from  primers . As show4+ concentration in the culture supernatant and lipid contents in the Δ12Ma and Δ12Mc overexpressing transformants were detected, and the results are displayed in M. circinelloides  encoded for the malic enzyme in cytoplasm (cME) were chosen for analysis since the cME was assumed to be important for the generation of the reducing power in oleaginous microorganisms [cme1 expression level was greatly increased in both gene overexpressing transformants, whereas cme2 was only up-regulated in Mc-D12MC  and three genes encoding for G6PDH  in M. circinelloides. As shown in 6pgdh2, g6pdh2, and g6pdh3 were up-regulated significantly in Mc-D12MC and Mc-D12MA.The growth results showed that drastic changes had taken place in response to the manipulation of Δ12-desaturase gene in transformants. To better understand the transcriptional response of fatty acid desaturation occurring, we extracted the total RNA and analyzed the transcript levels of several genes in these strains  during the fermentation process. The genes  [M. alpina and M. circinelloides to investigate possible ways to overcome obstacles in obtaining LA-rich microbial lipids.Oleaginous microorganisms can accumulate lipids that account for 20–80% of the CDW, depending on the species and cultures. Especially, when the nitrogen source is depleted, the excess carbon source is converted into a fatty acid and stored in tri-acylglycerol forms . The oleelloides . Howevery acids) . Thus, ty acids) . TherefoM. alpina and M. circinelloides shared physiological functions in the M. circinelloides WJ11. We also observed that overexpression of the Δ12-desaturease gene from M. alpina and M. circinelloides in the WJ11 strain caused an increase in lipid production and suppression of cell growth in the nitrogen limited broth in the early fermentation stage  than that in Mc-D12MA . Considering the significant increase in LA for the total fatty acid and the lipids in transformants cells, we can hypothesize that the overexpression of the Δ12-desaturease could increase the activities of key enzymes in the NADPH synthesis pathway and further increase LA production and lipid accumulation.To further investigate the underlying mechanism of Δ12-desaturase in improving LA production and lipid accumulation, the present work has studied the genes generating NADPH, the redox cofactor, which is a critical factor for the biosynthesis of valuable lipids in oleaginous cells . TherefoM. alpina and M. circinelloides. The data presented an improvement in the LA production and specific lipid accumulation. Therefore, Δ12-desaturease gene overexpression in M. circinelloides can be used to solve the blockage that occurs during high-value fatty acid synthesis. However, further work is required to better understand the mechanisms associated with promising lipid production in organisms to soon be used in the pharmaceutical industry.We demonstrated the development of a genetic manipulation strategy for improving LA production, associated with increased NADPH availability resulting from the overexpression of the Δ12-desaturease gene in


These results suggest that already-expressed memory impairment in aged male mice that depend on cyclic nucleotide signaling can be reversed by inhibition of PDE3. The reversal of age-related memory impairments may occur in the central nervous system, either through cilostazol-enhanced recall or strengthening of weak memories that otherwise may be resistant to recall.The nucleotide second messenger 3′, 5′-cyclic adenosine monophosphate (cAMP) and 3′, 5′-cyclic guanosine monophosphate (cGMP) mediate fundamental functions of the brain, including learning and memory. Phosphodiesterase 3 (PDE3) can hydrolyze both cAMP and cGMP and appears to be involved in the regulation of their contents in cells. We previously demonstrated that long-term administration of cilostazol, a PDE3 inhibitor, maintained good memory performance in aging mice. Here, we report on studies aimed at determining whether cilostazol also reverses already-impaired memory in aged male mice. One month of oral 1.5% cilostazol administration in 22-month-old mice reversed age-related declines in hippocampus-dependent memory tasks, including the object recognition and the Morris water maze. Furthermore, cilostazol reduced neuroinflammation, as evidenced by immunohistochemical staining, and increased glucose uptake in the brain, as evidence by positron emission tomography (PET) with 2-deoxy-2-[ A growing awareness of dementia and age-related cognitive and memory impairment highlights their impact on persons, families, and society, especially aging populations . Great eAplysia  are the only enzymes that degrade cyclic nucleotides including cAMP and/or 3′, 5′-cyclic guanosine monophosphate (cGMP) by breaking their phosphodiester bond . The inhCilostazol is currently prescribed as an antiplatelet agent for the treatment of chronic peripheral arterial occlusion  and inte18F]fluoro-d-glucose ([18F]FDG).In the present study, we examined whether cilostazol administration reverses already-established memory impairment in aged C57BL/6J mice. For this purpose, we administered cilostazol to 22-month-old C57BL/6J mice that exhibited extensive cognitive impairment , and theGuide for the Care and Use of Laboratory Animals (In Vivo Experiments (ARRIVE) guidelines , and also in accordance with  Animals . Here, widelines .The health of mice was monitored daily by animal technicians. In the rare cases when a mouse rapidly lost weight or became emaciated, it was killed humanely. We carefully considered the 3Rs . In the present study, we defined 3-month-old mice as young mice and 23-month-old mice as aged mice. . Mice we®  to monitor the mice during behavioral testing in the open field test, Morris water maze, and fear conditioning. Time® was also used to monitor and analyze the home-cage activity of the mice online. The Time® data acquisition system was used to control experimental devices and to analyze behavioral data.All apparatuses used in this study were obtained from O’Hara & Co., Ltd , unless specified otherwise. The open field test and the fear conditioning apparatus were installed in the sound proof rooms. We used the company’s video analysis and automation software Timead libitum until the end of the experiments. The feeding program for the control mice was the same, except the feed (CRF-1) contained no cilostazol (0% cilostazol). The control (0%) mice served as a non-drug control.Cilostazol  was mixed in the feed . After mice were kept with standard food in the vivarium for 20 months and 3 weeks, i.e., when they reached 22 months of age, they were assigned to one of three dose groups . We assigned mice in such a way that the mean body weight of each group was comparable. Mice were fed the food-cilostazol mixture g to collect serum. Serum cilostazol concentration was measured using a high-performance liquid chromatography system , according to a method reported previously , food, and water intake were measured in the home cages of the mice. Mice were individually housed in cages containing wood-chip bedding . After mice habituated to their new cage for 5 days, we recorded and calculated their spontaneous activity, and food and water intake on the sixth day . Food anMice were sequentially tested in the open field test, the object recognition task, Morris water maze task, and the Pavlovian fear conditioning task . After tTo examine the locomotor activity and anxiety-like behavior in a novel environment, mice were tested in the open field test as we described previously . BrieflyThe object recognition task is a well-established task to study responses of a rodent to spatial changes in the arrangement of objects and to novel objects . The tasSpatial memory was tested in the Morris water maze task . We testMice were tested to examine conditioned fear to both a specific cue and testing context, as described previously . BrieflyFor the cue-dependent fear memory tests, mice were placed individually into a new chamber to which they were previously unexposed. The chamber used for the cue-dependent memory test was different from the shocking chamber in perspective of brightness, texture, and tactile. In addition, odor stimulus was used in the cue-dependent fear memory test for additional experimental context discrimination. This procedure was used to separate out the contributions of cue-dependent fear memory from context-dependent fear memory; the tone was presented for 60 s. Preliminary experiments confirmed that the experience at short-term cue-dependent memory test (1 h) have no effect on long-term cue-dependent memory test (24 h). For the context-dependent fear memory test, mice were placed in the original shocking chamber in which they received the shocks, but for this test, they received no footshock. Throughout the experiments, freezing duration was measured and used as an index of fear . We usedAfter the completion of the fear conditioning task, half of the mice  from eacWe measured tissue density of microglia and astrocytes, both of which become activated in neuroinflammation . After t2O2 and 0.3% normal goat serum  in PBS for 10 min, followed by blocking of non-specific binding and permeabilization with 1.5% NGS and 0.3% Triton in PBS for 1 h. Blocked sections were then incubated overnight at 4°C with anti-ionized calcium-binding adapter molecule 1 (Iba1) antibody , a marker for microglia, or anti-glial fibrillary acidic protein (GFAP) antibody , a marker for astrocyte diluted in PBS containing 0.5% NGS and 0.3% Triton. Immunoreactions were visualized using an ABC kit (Vector Laboratories) and 3,3′-diaminobenzidine as a substrate.Brains were coronally sectioned at 30 µm thickness using a cryostat . Sections were washed and subjected to blocking of endogenous peroxidase with 0.3% H2. Three to four independent sections per mouse were analyzed, and the average counts were used as a representative value of either microglia and astrocytes.To quantify Iba1-and GFAP-positive cells in each section, digital images were captured by using the virtual whole-slide imaging system . The numbers of Iba1-and GFAP-positive cells in CA1, CA3, and dentate gyrus (DG) of the hippocampal formation and cerebral cortex were quantified separately with ImageJ Fiji . Nissl-s18F]FDG in only aged mice that received 0% or 1.5% cilostazol, as described previously . The PET image data were normalized with a [18F]FDG PET template . T1-weighted magnetic resonance images (MRI) of a representative mouse were also obtained with an ICON  to be used for superimposing PET-MRI images.FDG using PET. Increased [18F]FDG uptake reflects neuronal activity in the brain (18F]FDG uptake in the hippocampus (t (15) = 2.59, p < 0.05) and the whole brain (t (15) = 2.46, p < 0.05)  = 0.86, n.s.). These results exclude the potential differences of competitive inhibition on [18F]FDG uptake by circulating blood glucose  to aged mice for 1 month, and then we assessed the uptake of [he brain . The adm < 0.05)  compared < 0.05)  for the ake data . Severalake data . The reso uptake . Cilosta glucose .We previously reported that long-term cilostazol administration, starting from 13 months of age, prevented age-related memory impairment that normally manifests at 23 months of age in male C57BL/6J mice . An openversus a fine-grained representation about the locations of objects . Because antiplatelet agent aspirin is used as comparator in the clinical trial, the diverse role of direct and indirect effects on cilostazol-induced reversal of cognitive function will be revealed. Further experiments that include more quantitative measurements of the cerebral glucose metabolic rate (CMRglc)   are requPre-dementia phases such as cognitive frailty is thought to be potentially reversible and preventable until probable point when neuronal damage becomes fatal , howeverIn the present study, male mice were used based on our previous data on the detailed characterization of drug-free age-related functional changes in behavioral phenotype  and on t


Lactiplantibacillus plantarum 299v, and two indicator strains of Staphylococcus aureus 25923 and Listeriamonocytogenes 15313 were used in the study. The experiment consisted in applying a stress factor  to the tested LAB cells to investigate the in vitro properties such as hydrophobicity abilities (against p-xylene and n-hexadecane), auto-aggregation, co-aggregation with pathogens, and inhibition of pathogens adhesion to the porcine gastric mucin. The bacterial strains showed various hydrophobicity to p-xylene (36–73%) and n-hexadecane (11–25%). The affinity for solvents expanded with increasing thermal inactivation time. All LAB isolates were able to auto-aggregate (ranging from 17 to 49%). Bacterial strains subjected to 5 and 15 min of thermal inactivation had the highest auto-aggregation ability in comparison to viable and heat-killed cells for 30 min. The LAB strains co-aggregated with pathogens to different degrees; among them, the highest scores of co-aggregation were observed for L. monocytogenes, reaching 27% (with 15 min of heat-killed LAB cells). All LAB strains reduced the adherence of pathogenic bacteria in the competition test, moreover, heat-killed cells  were more efficient than viable cells. The properties of selected LAB strains as moderately heat-stressed forms analyzed in the study increased the prevention of colonization and elimination of pathogenic bacteria in the in vitro model of gastrointestinal tract. The thermal inactivation process may therefore preserve and modifies some characteristics of bacterial cells.The study aimed to evaluate the effect of thermal inactivation of potentially probiotic lactic acid bacteria (LAB) strains isolated from food on their ability to compete with pathogenic microorganisms. Five strains of LAB, previously isolated from food and characterized, one commercial reference strain of  L. monocytogenes is especially important among high-risk consumer groups. However, most consumers today are highly opposed to food additives, considering them harmful, even if they are unable to explain how they affect their health [Lacticaseibacillus, Lactiplantibacillus, Levilactobacillus, and many others, which were historically grouped within one genus named Lactobacillus [Despite the undeniable and significant advances in the food industry, the incidence of foodborne diseases is still on the rise in the European Union. In the year 2019, campylobacteriosis and salmonellosis were the most common zoonoses, and the highest number of deaths of 31) was reported for listeriosis [1 was repr health . Among tr health , they arbacillus . Accordibacillus . The appbacillus . Althougbacillus ,12,13,14bacillus ,16. The bacillus . While, bacillus . Howeverbacillus . Therefobacillus ,22. EvenThe use of postbiotics has several advantages over living organisms. The main argument for the safety of using inactivated cells of probiotic strains is the fact that such cells have lost their ability to proliferate . Other bListeria monocytogenes and Staphylococcus aureus. In addition, verification of how the heating time of LAB cells influences their selected biological properties that are important for adhesion capacity was investigated. To the best of the knowledge, this is the first study in which different heat-inactivation time of LAB strains was compared in terms of preserving their antimicrobial properties. The research is part of the current approach to the importance of providing more evidence to validate the beneficial effects of postbiotics for the future development of prophylactic or therapeutic agents as well as functional food or food additives.Therefore, the purpose of the presented study was to evaluate in vitro the five potentially probiotic LAB strains previously isolated from food as viable, and heat-killed forms, against two model pathogen microorganisms: Lacticaseibacillus casei O12, Lacticaseibacillus casei O16, Levilactobacillus brevis O22, Levilactobacillus brevis O24, and Lactiplantibacillus plantarum O20. The strains were isolated from traditional fermented food and molecular typing and in vitro probiotic properties of the strains were previously described [Lactiplantibacillus plantarum 299v was used as a reference strain. In this study, the strains were marked with the corresponding symbols: O12, O16, O22, O24, O20, and 299v, respectively.Five different LAB strains were used: escribed ,31. The All bacterial strains were kept in a mixture of de Man, Rogosa, and Sharpe (MRS) broth  (800 μL) and glycerol (200 μL) at −80 °C until further use. LAB strains were twice subcultured on MRS broth every 24 h before experimental use. A serial dilution of sterile peptone saline  in MRS agar  was conducted to determine the colony-forming units CFU/mL. Incubation was managed in anaerobic conditions for 48 h at 37 °C.Listeria monocytogenes strain (ATCC® 15313™), and Staphylococcus aureus strain (ATCC® 25923™) from the collection of American Type Culture Collection (ATCC). The following selective media was used to enumerate: Palcam Agar Base  for Listeria, and Baird-Parker Agar  for Staphylococcus. Singe colonies had been transferred to Nutrient broth . Cultivation was carried out at 37 °C for 24–48 h aerobically. Indicator strains used for evaluation of antimicrobial properties were g for 10 min. The supernatant was then decanted and the remaining pellet was suspended in 10 mL of PBS  solution . This action was repeated twice. Afterward, the pellet was resuspended in an appropriate volume of PBS solution and standardized to OD600 = 1.0 (2 × 108 CFU/mL) in the studies of hydrophobicity, auto-, and co-aggregation. Cultures with an OD600 value of 0.5 were prepared for the study concerning the ability to inhibit the adhesion of pathogens. The inactivation step was applied by placing the obtained solutions in the water bath at 80 °C, and then the appropriate volumes of suspensions were taken after 5, 15, and 30 min. Viable cells of LAB were also used in the study.Thermal inactivation was used as a fixation method to preserve the bacterial surface proteins unchangeable, as well as to kill bacteria cells entirely. For this purpose, cultured bacteria were precipitated by centrifugation in 5000 × 600 = 1, in the amount of 3 mL, were transferred to separate tubes. The samples were cooled to room temperature, and then 1 mL of n-hexadecane solvent  or 1 mL of p-xylene solvent  was added separately to each of the six tested strains. After the solvent was added, each sample was vigorously vortexed for 20 s. The samples were incubated for 20 min at room temperature. After incubation, the two phases were separated, and the aqueous phase was carefully removed, in the amount of 1000 µL was applied to a 24-well plate, and its absorbance at 600 nm in a spectrophotometer  was measured. The hydrophobicity percentage was calculated using the following formula:n-hexadecane/p-xylene, respectively.Bacterial cell surface hydrophobicity was assessed by measuring microbial adhesion to solvents (MATS) as described by Pérez et al.  with minData were expressed as a mean ± standard deviation which was derived from the hydrophobicity values of three independent experiments.The auto-aggregation assay for LAB strains was done according to the method of Kos et al.  with cerThe auto-aggregation assay was also carried out for pathogen strains to acquire the auto-aggregation percentages so that the obtained results could be used to calculate the co-aggregation percentages.Data were expressed as a mean ± standard deviation which was derived from the auto-aggregation values of six independent experiments.Coaggregation was performed according to Kos et al.  with somData were expressed as a mean ± standard deviation which was derived from the co-aggregation values of four independent experiments.L. monocytogenes and S. aureus cell suspensions were centrifuged 5000 × g for 10 min. The resulting pellets of bacterial cells were subjected to fluorescence staining by dissolving the pellet in 1 mL of CFDA dye (5-carboxyfluorescein diacetate)  at a concentration of 100 µmol/L. The cultures were incubated for 30 min at 37 °C. After this time, the cultures were centrifuged and washed 2 times in 5 mL PBS. Then 100 μL of viable or heat-killed LAB strains together with stained cells of pathogens suspension were added to the wells coated with mucin and incubated for 1h at 37 °C. Thereafter, the wells were washed two times with PBS buffer and adhered cells were recovered by treating each well with 200 μL of 1% (w/v) SDS (sodium dodecyl sulfate)  in 0.1 M NaOH solution for 30 min at 37 °C. At this time, the contents of each well were transferred in the same order to a black 96-well fluorescence plate. The fluorescence was measured at λex = 488 nm and λem = 538 nm in a spectrophotometer . Mucin with PBS addition without added bacteria was used as negative control and the absorbance value of this negative control was subtracted from the absorbance value of the samples.Adhesion assays (in vitro) were performed according to Moisés Laparra et al.  with somL. monocytogenes and S. aureus was constructed to estimate the number of mucin-bound bacteria (CFU/mL), from which the percentage of pathogen cells reduced was then calculated. Serial dilutions of the contents of each well were made in PBS . The solutions were applied in an amount of 200 µL to a 96-well plate and at wavelengths identical to the previous experiment, fluorescence was measured and standard curves were prepared.To analyze the results of inhibition of pathogen adhesion, a calibration curve for Percentage inhibition of pathogens adhesion was calculated as follows:Data were expressed as a mean ± standard deviation which was derived from the inhibition of pathogens adhesion values of six independent experiments.L. casei O12, L. casei O16, L. brevis O22, L. brevis O24, L. plantarum O20, L. plantarum 299v) and their interaction . In each of the studies, the effect of the heat inactivation time and the effect of the type of strain on potentially probiotic properties were subjected to a one-way analysis of variance (ANOVA). The comparison of post-hoc means was performed with Tukey’s range test. When the assumptions of a parametric test were not respected, Kruskal-Wallis, a nonparametric test, along with Dunn’s multiple comparison test was applied. p values less than 0.05 were considered statistically significant.The STATISTICA 13.3 Software  was used to perform the statistical analysis. All results  obtained in the study were statistically analyzed using a two-way ANOVA. The model included the effect of time of the heat inactivation  of tested bacterial strains  followed by n-hexadecane (11.00–24.73%).The LAB isolates showed different hydrophobicities in terms of the affinity to p < 0.001) and type of bacterial strains (p < 0.001) on the cell surface hydrophobicity against p-xylene was observed. The least affinity to p-xylene was observed for viable cells of commercial strain L. plantarum 299v (36.03%). Nevertheless, a significant (p < 0.05) increase in hydrophobicity was observed between viable cells and heat-killed cells for 5 and 30 min in the case of L. plantarum 299v. In addition, a significant increase (p < 0.05) in the affinity of bacterial isolates for p-xylene regarding viable cells was observed for strains L. brevis O22 (for a 30-min inactivation), L. casei O16 (for 15- and 30-min inactivation), and L. brevis O24 .A significant influence of heat inactivation time (n-hexadecane based on the heat inactivation time (p < 0.001) and type of bacterial strain (p < 0.001) were found. This effect was not seen in a combination of those two factors. When tested for cell surface properties using n-hexadecane, the highest hydrophobicity was determined for heat-killed cells for 30 min of the L. brevis O22 strain (24.80%). A significant increase of hydrophobicity (p < 0.05) in comparison to viable cells was detected for heat-killed cells (t = 15') of L. casei O16 strain (17.23%). In this case, a decrease in the hydrophobic properties of L. plantarum 299v cells was observed after exposing them to thermal stress. Anyhow, the result did not reach statistical significance.Significant differences in the cell surface hydrophobicity of bacterial strains against Six LAB strains exhibited auto-aggregation ranging from 17.08 to 48.87% after incubation at 37 °C, as shown in p < 0.001). The auto-aggregation values were in the range from 17.08% in 30 min heat-killed forms of commercial L. plantarum 299v strain to 38.59% in 15 min heat-killed forms of L. plantarum O20 strain. There was a trend that with one-hour incubation of bacterial cells, the greater ability to aggregate was demonstrated by cells that were quenched with high temperature for 5 min and 15 min (p > 0.05). On the other hand, treating bacteria cells with temperature for 30 min weakened their power to clump together (p > 0.05). The values of auto-aggregation that characterized all heat-killed forms for 30 min were lower than the control samples, except for the L. brevis O20 strain.After an hour of incubation, the results indicated that the heat inactivation time and type of bacterial strains separately significantly affected the auto-aggregation properties . The weakest auto-aggregation ability was demonstrated by heat-killed forms for 30 min of L. brevis O22 and O24 strains.After 24 h of incubation, the values of auto-aggregation fluctuated in the range from 24.13 to 48.87%. In this case, heat-killed forms for 5 and 15 min were also characterized by the greatest ability to form auto-aggregates. Heating the cells for 30 min reduced their auto-aggregation potential. The strain L. monocytogenes cells is shown in The co-aggregation ability of investigated LAB with Listeria monocytogenes. At time 0, heat-killed forms for 15 min of the L. casei O12 strain showed the highest co-aggregating ability (27.03%) with L. monocytogenes, whereas the lowest was noticed in the case of commercial strain L. plantarum 299v viable cells (6.69%). A significant increase (p < 0.05) in co-aggregation ability between viable and heat-killed cells was observed solely in commercial strain L. plantarum 299v. In other strains, the percentage of co-aggregation ability fluctuated insignificantly. The results for this incubation time indicate that the ability to form co-aggregates was influenced only by the strain type (p < 0.05).All tested strains showed some co-aggregation properties with the foodborne pathogen of L. casei O12 strain exhibited the best co-aggregation properties after an hour of incubation, particularly 15 min heat-killed forms of the L. casei O12 strain showed the highest co-aggregation ability (12.72%), which was higher (p < 0.05) than the percentages of the co-aggregation of L. plantarum O20 strain.All treatments of the Listeria monocytogenes after 24 h of incubation. This ability was thermal inactivation time-specific (p < 0.05), and the percentages fluctuated. Significant differences in the co-aggregation ability between heat treatments within the two strains were noted. Co-aggregation of 15 min of heat-killed forms (12.74%) in comparison to 5 min of heat-killed forms (1.31%) and 30 min (1.40%) of the L. casei O16 strain was greater (p < 0.05). On the other hand, the ability of viable cells of L. brevis O22 strain (10.20%) to co-aggregate with the pathogen was greater (p < 0.05) than that of cells of the same strain but exposed to thermal shock for 5 min (1.13%) and 30 min (0.83%).All tested viable and heat-killed forms showed some co-aggregation ability with Staphylococcus aureus and viable and heat-killed forms of investigated LAB stains is indicated in The co-aggregation between S. aureus seems to be species-dependent (p < 0.001). L. casei O12 and O16 were the best aggregating strains, while L. brevis O22 and O24 were the worst. No unequivocal tendency was found in the case of the tested L. plantarum species. The probiotic strain L. plantarum 299v co-aggregated successfully, while L. plantarum O20 showed a poor ability to co-aggregate. In this case, a negative co-aggregation capacity between the strains of L. plantarum O20, L. brevis O22, and L. brevis O24, and the pathogen S. aureus was observed.It can be observed that the ability to co-aggregate with L. casei O12 and L. casei O16 was 14.20 and 16.94% respectively, and co-aggregation of thermal inactivated cells for 15 min of corresponding strains amounted to 25.06 and 25.57% respectively.At time 0, significant differences were observed between viable and heat-killed forms, consisting of a significant increase in co-aggregation in favor of the latter. Co-aggregation of viable cells of Lactobacillus strain after 1-h co-aggregation. The co-aggregation ability of heat-killed cells for 5 and 15 min of the L. casei O12 strain  was significantly lower than the coaggregation of viable cells (8.40%) and those heated for 30 min (8.85%). Similarly, the co-aggregation ability of viable cells of the L. casei O16 strain (8.23%) appeared substantially higher than the co-aggregation ability of all the bacterial heat-killed forms of this strain . A significant relationship (p < 0.05) was also observed between viable cells of the L. brevis O22 strain (7.08%) and those exposed to heat stress for 5 min (−14.57%).Also, significant differences were found within viable and heat-killed cells of the p < 0.05) in the co-aggregation ability percentage was reported only between heat-killed forms for 5 (19.51%) and 30 min (8.97%) of the L. casei O16 strain.The percentages of co-aggregation ability varied after incubation of cells for 24 h. A significant decrease (In p < 0.001) and type of bacterial strain (p < 0.001) significantly affected the inhibition of pathogens’ adhesion to the mucin.The results indicate that heat inactivation time (p < 0.05) between the inhibition of adhesion of L. monocytogenes to porcine gastric mucin by viable and heat-killed forms of the strains L. brevis O24 and L. plantarum O20 were noted. In the case of L. casei O16, L. brevis O22 strains, and commercial L. plantarum 299v strain, exposure of the cells to high temperature reduced the inhibition of pathogen adhesion to the porcine gastric mucin. The difference was significant (p < 0.05) for the L. brevis O22 strain.Significant differences (L. brevis O24 and L. casei O16 heat-killed forms inhibited the adhesion of S. aureus to a greater extent (p < 0.05) in comparison to the control sample. This effect was seen when heat-killed cells for 30 or/and 15 min were used. The weakest reduction of adhesion by heat-killed cells was observed in the case of L. brevis O22—about 56.23%, compared to viable cells (p > 0.05).It was also observed that To inactivate bacterial cells and preserve their surface structures, different fixation methods have been demonstrated in the literature, such as inactivation by chemical agents, heat, sonication, and UV irradiations . Each stCell hydrophobicity is an important attribute for probiotic bacteria, as well as for heat-inactivated cells due to its correlation to adhesion ability. The cell’s ability to adhere to the intestinal epithelium is considered a prerequisite for colonization of the human gastrointestinal tract so probiotics or postbiotics can exhibit a range of beneficial effects such as the exclusion of enteropathogenic bacteria ,39. It iThe adhesion of tested bacterial strains to solvent assay was conducted to investigate the features of the physicochemical cell surface . The MATp-xylene describes bacterial cell surface hydrophobicity. On the other hand, n-hexadecane was used as a nonpolar solvent having intermolecular attraction comparable to that of chloroform. The hydrophilic and hydrophobic features are attributed to polysaccharides and proteins on the bacterial surface [Adherence to a solvent  surface . Moreove surface . TherefoLactobacillus acidophilus CBT LA1 cells to thermal stress at 80 °C for 10 min, increase the hexadecane hydrophobicity by 7.26%, and the difference was significant (p < 0.05) [n-octane hydrophobicity of the Lacticaseibacillus rhamnosus GG cell surface was observed. In the conducted study the hydrophobic cell surface was shown by high adherence to p-xylene followed by n-hexadecane. High affinity to p-xylene represents an advantageous hydrophobic ability. On the other hand, a lower percentage of bacteria adhering to a nonpolar solvent such as n-hexadecane may be indicated that the tested strains had low hydrophobicity. Also, tested LAB strains still simultaneously showed some affinity to p-xylene and n-hexadecane hydrocarbons, suggesting a high complexity of the cell surface. The existing distinctions in the present study might be a consequence of varied surface molecules such as the presence of exopolysaccharides (EPS) and specific surface proteins, additionally influenced by the effect of high inactivation temperature.In the present study, the cell surface hydrophobicity varied with the isolates tested. In the profuse majority of cases, the affinity for solvents expanded with increasing thermal inactivation time, and certainly the values for heat-killed cells were greater than those for viable cells with some significant differences . Similar < 0.05) . Collado < 0.05)  have fou < 0.05) . Opposit < 0.05) , where aBacteria are keen on existing in consortia, sticking together and/or to other bacterial cells, and adhering either to surfaces. Bacterial aggregation between cells of the same strain in a nonspecific way is called auto-aggregation. This process consists of the reaction of bacterial strains physically with each other and precipitates from static liquid suspension . The abip < 0.05).The results obtained in the conducted study showed that all viable and heat-killed isolates have comparable auto-aggregation ability ranging from 17.08 to 48.87% with the best results obtained after 24 h of incubation. Many other studies observed the increase of auto-aggregation ability of LAB cells proportionally to time, with the highest values after 4 h , 5 h 3838,54,55,Since moderately heat treated cells appeared to have higher auto-aggregation ability than viable cells, these properties are likely to contribute to the higher adhesion rate to intestinal epithelial cells for colonization. Consequently, tested in the present study heat-killed cells of LAB strains or postbiotics from these bacterial cells can probably exert beneficial effects such as modulation of the immune response and development of a gut barrier against pathogens to a similar or greater extent than viable cells. However, this thesis should be verified during further studies.Co-aggregation is a process by which genetically different bacterial cells become attached via specific molecules forming a complex of multi-species biofilms . The abiL. monocytogenes and S. aureus, the highest percentages of co-aggregation ability were obtained directly after combining the cells of both strains (LAB versus pathogen), with a sedimentation time of 0. It was shown that the ability to form co-aggregates by the tested strains was greater in the case of L. monocytogenes compared to S. aureus. In the previous study [L. monocytogenes and the lowest against S. aureus. This study seems to maintain this relation.In the conducted study, measurements of the number of bound tested LAB cells to pathogenic bacteria were made at the beginning of the experiment, after an hour, and after 24 h of incubation. Regarding co-aggregation with us study  accordinL. monocytogenes and S. aureus. Several significant differences in favor of the heat-killed LAB cells were noted, but cannot be applied to all tested strains. It is also intractable to refer to the results of research by other authors since there are limited data available. Tareb et al. [L. rhamnosus 3698 and L. farciminis 3699 with the feed/food chain pathogens: Escherichia coli, Salmonella spp., Campylobacter spp. and Listeria monocytogenes after 24 h of incubation at 20 °C. The ability of Campylobacter strains to co-aggregate with probiotic strains was significantly better than that of other pathogenic strains tested (p < 0.05). Additionally, irrespective of the lactobacilli’s physiological status (viable or killed), the coaggregation ability remained similar. On the other hand, in the present study, the strain-specific co-aggregation ability was found. Similarly, Kim et al. [Lactobacillus acidophilus 11869BP heat-killed cells in comparison to viable cells had a higher co-aggregation ability with Salmonella Typhimurium. LAB viable cells displayed 43.5% co-aggregation ability, while heat-killed LAB cells displayed 55% ability [Lactobacillus acidophilus was able to co-aggregate with Fusobacterium nucleatum. However, after 15 min and 30 min of incubation the viable cells bound to the F. nucleatum to a slightly greater extent than the heat-killed cells (p > 0.05) [It is difficult to estimate unequivocally that thermal inactivation improved the co-aggregation ability with b et al.  examinedm et al.  indicate ability . Additio ability  demonstr > 0.05) , which wL. monocytogenes adhesion, and the S. aureus adhesion inhibition by tested bacterial strains showed high variability. However, heat-killed cells  were more efficient than viable cells. Whatever the mechanism of exclusion, both heat-killed and viable cells of LAB strains prevented efficiently the adhesion of the pathogens to mucin.The protective role of probiotic bacteria against pathogens in gastrointestinal conditions and the underlying mechanisms is the subject of scientific interest . AdhesioC. jejuni CIP 70.2T was improved, especially for heat-killed forms of L. farciminis 3699 (from 15 to 70%). Also in our previous study [E. coli, S. aureus, L. monocytogenes, and Salmonella) with viable and heat-killed (80 °C per 20 min) cells of Lactobacillus strains were executed (including strains used in the conducted study). It was observed that inhibition of adhesion to Caco-2 cells was higher when cells of indicator strains were treated by viable than heat-killed cells of LAB. Although, in most samples, the effect did not differ significantly (p > 0.05). The inhibition effect was elevated in the case of E. coli, Salmonella, and L. monocytogenes, and demoted in the case of S. aureus. However, the results observed by other authors are ambiguous [In accordance with the results of conducted study, it has been previously reported that certain thermal inactivated cells of probiotics strains or postbiotics can inhibit mucosal or epithelial adhesion of different pathogens. Results of the study of Tareb et al.  demonstrus study  competitmbiguous ,68,70.As adhesion inhibition is influenced by cell-surface properties, in the conducted study the question of whether or not the viability of bacteria is an essential criterion was considered. Inhibition adhesion of pathogens by heat-killed LAB cells in the present study has been demonstrated, so the elucidation of improvement of inhibition adhesion by heat-killed cells is desirable. Thermal inactivation of cells may lead to the rupture of their walls, thereby releasing cytoplasmic contents, like DNA, as well as components of their walls, such as peptidoglycans, lipoteichoic acids, or heat-labile pili. The released bacterial components (postbiotics) could be responsible for the adhesion inhibition of pathogens ,72. It hThe conducted study provided new information regarding the use of viable or heat-killed forms of selected LAB strains. The process of fixation with heating did not cause loss of the tested probiotic properties of LAB cells. It was observed that the modulation of these features was bidirectional, but did not result in their disappearance. Thermal inactivation for 5 and 15 min was a particularly prospective fixative, strengthening adhesion and aggregation properties of selected bacterial cells.Although the strains used in this study were previously tested for their probiotic properties, however, these properties are related to the viability of the bacteria cells. Postbiotic metabolites obtained after heat inactivation of culture offer wider possibilities of using such bacteria. Therefore, in the view of provided data, it can be concluded that the postbiotics of tested LAB strains exhibited a good ability to reduce the adhesion in vitro of some pathogenic bacteria to porcine mucin. Moreover, the abilities of auto-aggregation, co-aggregation with pathogens, and cell surface hydrophobicity were rather strain-specific characteristics of these microorganisms. Due to their cell-surface properties and antimicrobial activity, especially their anti-listeria activity, the selected postbiotics of LAB strains isolated from foods can be used as possible protective agents. As a limitation of the study, still unclear mechanism of postbiotic action should be underline. Therefore, further studies are needed to explain the molecular basis of changes in bacterial features as a result of thermal inactivation.The properties of the heat-killed probiotic cells to remain abilities of pathogen adhesion inhibition could enable those strains to be kept functional in unfavorable conditions. Future studies should focus on development and tested of the manufacturing process in purpose to offer more stable products characterized by identical properties such as original, probiotic formulations.


Background and Objectives: Injuries associated with electric bikes (E-bikes) and powered scooters (P-scooters) have increased yearly worldwide. We aimed to evaluate the impact of pre-existing comorbidities on the probability of hospitalization for injuries in riders of E-bikes and P-scooters. Materials and Methods: A retrospective cross-sectional study design was used. The cohort included patients referred to the emergency department (ED) of a tertiary medical center in 2014–2020 for injuries sustained while riding an E-bike or P-scooter. Data were collected from the medical files on demographics, clinical characteristics including pre-existing comorbidities and permanent use of medications, and injury characteristics. Findings were compared between patients referred for hospitalization from the ED and patients discharged home. Results: Of the 1234 patients who met the inclusion criteria, 202 (16.4%) had a prior medical condition and 167 (13.5%) were taking medication on a permanent basis. A significant relationship was found between hospitalization and having a medical condition (𝜒2(1) = 9.20, p = 0.002) or taking medication on a permanent basis (𝜒2(1) = 6.24, p = 0.01). Hospitalization for injuries was more likely in patients with a comorbidity (27.8%) than those without a comorbidity (15.5%), and in patients who were on permanent drug therapy (22.2%) than in patients who were not (12.9%). Surprisingly, anticoagulant intake specifically had no effect on the probability of hospital admission. Conclusions: Patients with comorbidities have a higher incidence of hospitalization for E-bike- and P-scooter-associated injuries. Therefore, physicians may take into account comorbidities for the effective management of this patient group’s injuries. The increasing use of electric bicycles (E-bikes) and powered scooters (P-scooters) in recent years has been accompanied by associated injuries ,2. StudiElectric vehicle injuries mainly occur in the extremities and maxillofacial areas ,11. A reIt is widely recognized that advanced age and comorbidities have a crucial impact on the probability of requiring medical care and hospital admission in cases of injury . MoreoveThe aim of the present study was to evaluate the impact of pre-existing comorbidities and the permanent use of medication, with an emphasis on anticoagulants, on the rates of hospitalization for injuries sustained by riders of E-bikes and P-scooters.A retrospective, cross-sectional study was conducted in the emergency department (ED) of a tertiary medical center from January 2014 to March 2020. ED referrals of patients that were injured while riding P-scooters and E-bikes met the inclusion criteria of the study. The ED database was first searched using the keywords “electric scooter” or/and “electric bike” and/or “powered scooter” or/and “powered bike” and “injury/injured”. The files of the patients identified were then further reviewed, and those who were found to have been actually involved in an E-bike or P-scooter accident during the study period and for whom sufficient data were available were included in the study.The following data were collected from the medical files: demographics, comorbidities, intake of permanent medications, use and specific type of anticoagulants, reason for prescription of anticoagulants, bleeding/hemorrhage, bleeding organs, need for hospitalization for current E-bike or P-scooter injury, and length of current hospitalization. The findings were compared between patients who were hospitalized and patients who were discharged home from the ED. An injury requiring hospitalization was defined as severe. The study protocol was approved by the Helsinki Committee of Rabin Medical Center .p-value lower than 5%.The data were analyzed using SPSS statistical software, version 25 . Continuous variables were summarized by means and standard deviations and discrete variables were depicted as frequencies. Chi-square test was used for univariate analysis. Owing to the non-normal distribution of the continuous variables, the nonparametric Mann–Whitney test was used to compare independent samples. Significance was defined as a A total of 1234 patients met the inclusion criteria; all of the patients were referred to the ED of “Rabin medical center” in Petah Tikvah, Israel regarding injuries associated with E-bikes or P-scooters during the study period. The full descriptive statistics of the cohort are shown in n = 30, 2.4% of the cohort). Eighteen patients (1.46%) reported being on permanent anticoagulant therapy. Aspirin, a salicylate antithrombotic agent, was the most common anticoagulant used .Permanent medications were being used by 167 patients (13.5%). Medications for diabetes were the most common prescribed (The E-bike/P-scooter injury was associated with hemorrhaging in 131 patients (10.6%) and with a bleeding organ in 124 patients (10.1%). The most common sites of bleeding were the eyes; intra-bulbar hemorrhage and the surrounding soft tissue, including the eyelids, in 28 patients (2.3%).2(1) = 9.20, 𝑝 = 0.002). Patients with comorbidities were more likely to be hospitalized (27.8%) than patients without comorbidities (15.5%). There was also a direct relationship between intake of permanent medications and hospitalization (𝜒2(1) = 6.24, 𝑝 = 0.01), with patients who were on permanent drug therapy having a higher likelihood of hospitalization (22.2%) than patients who were not (12.9%).Univariate analyses were used to test the association of each independent variable with hospitalization for injuries associated with E-bikes and P-scooters. The results are shown in 2(1) = 11.22, p = 0.001) and with bleeding from an organ (𝜒2(1) = (1) = 13.38, p = 0.001). Patients with hemorrhaging or bleeding were more likely to be hospitalized (21.1%) than patients without hemorrhaging or bleeding (9.8%), as were patients with a bleeding organ (21.1%) compared to patients without a bleeding organ (9.8%). The use of anticoagulants was not associated with an increased incidence of hospital admission.Hospitalization was significantly associated with hemorrhaging or bleeding  relative to the number reporting use of any prior permanent medications . Moreover, our evaluation of the direct impact of anticoagulant use on head injuries was limited because of a lack of data on head injuries specifically and helmet use.In the medically compromised population, impaired mobility, sight, and stability often lead to injuries due to falls and traffic accidents. On the basis of studies describing the impact of anticoagulant use on brain injuries ,14,15,16Diabetes mellitus is a widespread chronic disease that leads to impaired wound healing and can be classified as type 1 or type 2 ,18. Type17. Furthermore, hyperglycemia causes the delayed release of oxygen from the hemoglobin complex, leading to oxygen and nutrient deficiency in the wound area [At the cellular level, hyperglycemia interferes with wound healing owing to its adverse effects on neutrophil, lymphocyte, and mast cell function. It hampers red blood cell permeability and decreases blood flow through small vessels at wound surfaces resulting in impaired chemotaxis and phagocytosis . A recenund area .n = 36; 2.9%), and diabetic medications were the most common permanent drugs used . We assume that the impaired wound healing due to diabetes results in hard- and soft-tissue wound complications. As a consequence, the injuries sustained are more severe and require in-hospital management. This is supported by the significant relationship found here between having a medical condition and hospitalization (p = 0.002).At the tissue level, studies found diabetes to be significantly associated with an increased risk of insufficient hard-tissue  and softIn conclusion, the present study shows that the presence of underlying comorbidities and the long-term intake of medications increase the probability of hospitalization for injuries in E-bike and P-scooter accidents. The medical staff of the ED should be alert to the need to take the presence of comorbidities and permanent medication intake into account in predicting the severity of injury and the management of this patient group, as reported in the current study as a predictor for hospital admission.